A rare, uniformly lethal, undifferentiated midline carcinoma of young people is defined by the t(15;19)(p13.1;q13), whose product is the BRD4-NUT1 fusion oncogene. The molecular mechanism of BRD4-NUT1 carcinomas (BNCs) is unusual for a carcinoma, but resembles leukemias and sarcomas. Study of leukemic fusion oncogenes has provided important insights into oncogenic pathways, and normal hematopoietic development. These insights have led to the development of rational molecular therapies. The predicted BRD4-NUT1 fusion protein contains two BRD4-derived bromodomains, which bind acetylated histones in regions of transcriptionally active chromatin, and two NUT1-derived transcriptional activation domains. We have found that withdrawal of BRD4-NUT1 from BNC cell lines results in striking changes in vitro and in vivo that are consistent with the induction of squamous differentiation, suggesting that BRD4- NUT1 contributes to carcinogenesis by blocking terminal epithelial differentiation. These observations have led us to formulate two hypotheses, which serve to organize the studies proposed in this application: 1. BRD4-NUT1 blocks differentiation by altering the transcription of critical regulator(s) of epithelial cell differentiation. 2. This alteration in transcription is enacted by the interaction of NUT1 with specific protein partners. An underlying subtext is that these studies are likely to elucidate pathways that are vital to epithelial cell differentiation, proliferation, and neoplasia. In doing so, it is hoped that new rational therapeutic strategies relevant to BNCs and possibly other epithelial tumors will emerge. Based on these preliminary data and hypotheses, we propose the following three specific aims: 1. To identify the domains of BRD4-NUT1 that are necessary and sufficient to block differentiation. 2. To identify the BRD4-NUT1-interacting proteins that execute the downstream events that lead to the blockade of differentiation. 3. To identify the transcriptional targets of BRD4-NUT1 that are responsible for the blockade of differentiation in BRD4-NUT1 carcinoma cells.